This invention relates to the use of recombinant DNA techniques to produce heteropolymeric proteins.
Various polypeptide chains have been expressed, via recombinant DNA technology, in host cells such as bacteria, yeast, and cultured mammalian cells. Fiddes et al., Nature 281:351, 1979, and Fiddes et al., Nature 286:684, 1980, describe the cloning of, respectively, the xcex1 and xcex2 subunits of human choriogonadotropin (hCG).
Kaname U.S. Pat. No. 4,383,036 describes a process for producing hCG in which human lymphoblastoid cells are implanted into a laboratory animal, harvested from the animal, and cultured in vitro; accumulated hCG is then harvested from the culture.
In general, the invention features, in one aspect, a biologically active heteropolymeric protein composed of a plurality of subunits, both subunits being synthesized in a single cell having an expression vector containing heterologous DNA encoding the subunits. In related aspects the vector is autonomously replicating (i.e., not integrated into the chromosome of the host cell).
In preferred embodiments, the protein is synthesized by a eukaryotic cell, and the protein is modified post-translationally, most preferably by glycosylation; and the protein is a secreted protein such as a hormone, most preferably a fertility hormone such as hCG, luteinizing hormone (LH) or follicle stimulating hormone (FSH); or the hormone thyroid stimulating hormone (TSH). In other preferred embodiments the protein is an ungulate fertility hormone selected from LH and FSH; the ungulate being chosen from a horse, pig or cow.
In another aspect, the invention features a cell, containing a first expression vector, which cell is capable of producing a biologically active heteropolymeric protein that is encoded at least in part by the vector. In preferred embodiments, a second expression vector (which may be autonomously replicating) encodes a second portion of the protein or at least two subunits of the protein are encoded by a single expression vector; the protein is hCG or human luteinizing hormone (LH), or an ungulate fertility hormone selected from LH and FSH; the vector is a replicating virus or a plasmid; the cell is a monkey or mouse cell; transcription of the different subunits is under the control of the SV40 late promoter; transcription of the xcex1 subunit of the protein is under the control of the SV40 early promoter and transcription of the xcex2 subunit is under control of the mouse metallothionein promoter, or transcription of both subunits is under the control of the mouse metallothionein promoter; and the expression vector which includes the mouse metallothionein promoter also includes at least the 69% transforming region of the bovine papilloma virus (BPV) genome.
In another aspect, the invention features an expression vector (which may be autonomously replicating) including two genes encoding two different heterologous proteins, the genes being under the control of two different promoters, most preferably a metallothionein promoter and a BPV promoter; the use of different promoters advantageously minimizes the possiblity of deleterious recombinations.
In preferred embodiments of the above aspects the vector comprises at least a part of a DNA sequence chosen from the DNA sequences shown in FIG. 11, 12, 13, 18, 20, 21, 23, 27 or 32; and the fragment in the vector is chosen from those fragments deposited in cells in the N.R.R.L. with accession number B18124, B18125, B18126, B18127, B18128, B18139, B18140, B18131, or B15793.
As used herein, xe2x80x9csubunitxe2x80x9d refers to a portion of a protein, which portion, or homologue or analogue thereof, is encoded in nature by a distinct mRNA. Thus, for example, a heavy chain and a light chain of an IgG immunoglobulin are each considered a subunit. Insulin, on the other hand, is composed of two chains which are not considered subunits, because both are, in nature, encoded by a single mRNA, and cleavage into two chains naturally occurs only after translation.
The term xe2x80x9cexpression vectorxe2x80x9d refers to a vector which includes heterologous (to the vector) DNA under the control of control sequences which permit expression in a host cell. Such vectors include replicating viruses, plasmids, and phages. The term xe2x80x9cheterologous DNAxe2x80x9d is used to refer to DNA which does not naturally occur adjacent to any other DNA which is also being referred to.
The invention permits the production of a biologically active heteropolymeric protein from a single culture of transformed cells. We have found that when the xcex1 subunits of a heteropolymeric protein are produced alone they are modified by the host cell. This modification prevents recombination of the xcex1-subunit with the xcex2-subunit. Thus, biosynthesis of an active gonadotropin (i.e., xcex1-and-xcex2-subunits linked by ionic bonds) cannot occur in genetically engineered cells unless a system is used to produce both within a single cell so that combination of the subunits occurs prior to post-translational modification. This invention describes and provides the technology required for the establishment of stable cell lines transfected with foreign DNA that encodes for the biosynthesis of balanced amounts of both xcex1 and xcex2-subunits. These cell lines allow expression of biologically active dimeric glycoprotein hormones. The system allows production of proteins, in a single culture, which undergo, in the culture, post-translational modification, e.g. glycosylation and proteolytic processing, for activity or stability.
The use of autonomously replicating expression vectors prevents undesirable influence on the desired coding regions by control sequences in the host chromosome.
Other advantages and features of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
We turn now to the preferred embodiments of the invention, first briefly describing the drawings thereof.